Bioavailability and toxicity of dietary nickel and zinc to the waterflea Daphnia magna

Zinc (Zn) and nickel (Ni) are metals that have been increasingly mined, manufactured and employed in various applications during the past decades. This has worldwide resulted in a discharge that caused elevated levels in freshwater ecosystems. Policy makers have been urged to develop regulations to minimize the environmental risks related to Zn and Ni pollution, among other metals. Current environmental quality standards for metals, representing concentrations that pose an acceptable risk for aquatic communities, are derived on the basis of their bioavailability and not on total metal concentrations. This concept represents the principle that the toxicity of a metal is not only dependent on its total concentration, but also on the physico-chemical characteristics of the surrounding water. Bioavailability has been integrated in metal risk assessment procedures by the Biotic Ligand Model (BLM). For freshwater organisms, this model is developed from the basic assumption that the toxicity of a metal is the result of interaction with and uptake via ionoregulatory channels in the gills. However, the relatively recent discovery that dietary metals can also be toxic was expected to restrict the applicability of the BLM for several reasons. Firstly, it was assumed that this could result in an underestimation of the real exposure levels of metals, and that this in turn would end in the derivation of environmental metal standards that are not sufficiently protective. Secondly, this could also question if the mechanistic background of the BLM is still representative for the true mechanisms of metal exposure, since an extra exposure route would imply that the model assumptions are not valid anymore. Up to now, however, dietary metal exposure is still not considered explicitly in most existing metal regulations and risk assessment procedures. For various metals and aquatic species there is much uncertainty if there is relevance for this exposure route under realistic contamination levels. Also, a refinement of the BLM will only be possible until the links between dietary metal exposure and effects are better understood and quantitative methods are developed for linking metal exposure to toxicity. The freshwater flea Daphnia magna is a planktonic crustacean which occupies a central position in freshwater food webs. Its feeding and nutrition are important to both the impact on bacterial and algal populations, as well as the conversion of these micro-organisms into body mass used as food for predators. It has been extensively used as a model species for bioavailability studies and for the development of BLM‟s predicting the toxicity of, among other metals, waterborne Ni and Zn. At the moment this study was started, however, the knowledge on dietary Ni and Zn toxicity was scarce. In chapter 2 we studied if there is relevance for dietary Ni toxicity to D. magna under realistic exposure levels. The green alga Pseudokirchneriella subcapitata was pre-exposed to Ni, encompassing the EC50 for chronic waterborne Ni exposure on D. magna, and used as food during a 21-day chronic bio-assay. A significant accumulation of dietborne Ni in D. magna, i.e., between 49.6 to 72.5 μg Ni/g dry wt, was observed when they were fed with diets containing between 85.6 and 837 μg Ni/g dry wt. This was paralleled by a significant reduction of reproduction (by 33.1%) and growth (by 9.1%). A variety of mechanisms may have been involved in these effects, including altered resource allocation or targeted reproductive inhibition. No unambiguous conclusions could be drawn, however, since these exposures were also accompanied by an altered availability of phosphorus (P) and essential omega 3 polyunsaturated fatty acids (ω3-PUFA‟s). There was no conclusive evidence that nutritional quality shifts could have contributed to the toxic effects, but it is clear that the use of diets invulnerable to metal-induced quality shifts is indispensable for a sound study of the real impact of dietary Ni exposure and its toxicity mechanisms. This problem was circumvented in chapter 3. Ni-contaminated liposomes were used as an alternative dietary Ni carrier, invulnerable to nutritional quality changes. These were produced by the hydration of phosphatidylcholine with Ni-contaminated solutions above the transition temperature of this phospholipid. Particle sizes were shown to be appropriate to be captured by the filtration apparatus of D. magna. In a first experiment, daphnids were fed with a mixture of Ni-laden liposomes and control algae as a nutrient supply. Ni bioaccumulation up to 11.9 μg/g dry wt after 7 days and 20.4 μg/g dry wt after 14 days caused a significant inhibition of reproduction and growth. It was suggested that this was mediated by a reduced nutrient absorption during the first 7 days, and additionally by a reduced algal ingestion rate beyond 7 days of exposure. A simultaneous experiment was performed, using a mixture of Ni-contaminated algae (cfr. chapter 2) and control liposomes as a diet. D. magna accumulated Ni to a similar degree as the LOEC when the algae had been pre-exposed to at least 133 μg Ni/L of bioavailable Ni (Ni2+), which is similar to the EC50 of waterborne Ni exposure (115 μg Ni2+/L). This illustrated the consequences for the use of the BLM in regulatory assessments, which may need to include the dietary toxicity pathway in this range of dissolved Ni concentrations. There was no evidence that nutritional quality shifts could have affected daphnids growth, but it is very likely that the impairment of reproduction at toxic exposure levels of Ni was also partly the result of reduced α-linolenic acid levels. The integration of the dietary Ni pathway in the BLM could provide more mechanistic knowledge to predict the effects of chronic Ni exposures. It was expected that waterborne and dietary Ni bioaccumulation could be a good predictor of toxicity, but no knowledge existed on the importance of and potential interaction between both pathways during a simultaneous exposure. In chapter 4, a stable isotope tracer experiment was worked out to investigate the 7-day bioaccumulation of waterborne and dietary Ni at increasing concentrations in the water (0, 235 and 470 μg Ni/L) and at two pH levels (pH 6.4 and 8.2). Total Ni burdens in the daphnids evolved from 41.9 to 110.4 μg/g dry wt at pH 6.4 and from 26.4 to 58.5 μg/g dry wt at pH 8.2. Waterborne Ni uptake was significantly correlated to the concentration in the water, and was always the dominant source of uptake, contributing for at least 84% to the total amount of Ni present in the organisms. Important to note was that the uptake of dietary Ni also increased at elevated concentrations in the water, up to 12.4 and 9.1 μg Ni/g dry wt at pH 6.4 and 8.2, respectively. This synergy was important from a toxicological point of view. The bioaccumulation of dietary Ni in D. magna matched the LOEC for 7-day growth at 470 μg Ni/L, or 115 μg Ni2+/L in the modified M4 medium (chapter 3). This concentration was, however, too low to generate toxic Ni burdens in algae if these would be applied as a Ni-contaminated diet during diet-only exposures. This implied that the contribution of dietary Ni uptake to the toxic effects of a combined exposure may not be neglected. The occurrence of synergy could also explain why dietary Ni accumulation did not increase with increasing pH, although the Ni concentration in the food was significantly higher. Further research should be performed to elucidate the relation between Ni uptake and toxicity during a simultaneous exposure to waterborne and dietary Ni. At the moment this research was started, the implications of dietary Zn exposure had gained far more attention than Ni. With a view to refine the BLM for chronic Zn exposure, it was investigated if an unambiguous relation could be drawn between Zn bioaccumulation and toxicity of each exposure route. In chapter 5 we investigated the hypotheses (1) that dietary Zn toxicity is the result of a selective accumulation in reproductive tissues, and (2) that waterborne and dietary Zn accumulation and toxicity are additive. D. magna was exposed to Zn following a 5 x 2 factorial design, with 5 waterborne Zn concentrations (0, 80, 170, 250 and 340 μg Zn/L) and 2 dietary Zn levels (50 and 496 μg Zn/g dry wt). Tissue-specific Zn accumulation was quantified by means of Synchrotron Radiation based Confocal X-ray Fluorescence. Waterborne Zn exposure was optimal at 80 μg/L, whereas organisms suffered from deficiency and toxicity at lower and higher concentrations, respectively. Dietary Zn reduced reproduction by 28% and selectively accumulated in the eggs. These were probably provided when still present in the ovaries, which was in line with the first hypothesis. Dietary Zn was less toxic at higher Zn concentrations in the water, which could be attributed to a lower feeding rate resulting from waterborne Zn toxicity. Accordingly, no significant bioaccumulation could be detected in this concentration range. Both observations confirmed the second research hypothesis. This has obvious implications for the BLM, since the importance of dietary Zn exposures fades out at waterborne Zn concentrations that are shown to be toxic. For this reason we concluded that the BLM used in risk assessment exercises provides adequate protection against Zn exposure, and thus could take away the main concern of regulators with respect to the degree the current BLM should be refined. The complete absence of dietary Zn toxicity in the control treatment was not expected, and in contrast with previous studies dealing with dietary Zn exposure. We hypothesized that a variability of the nutritional quality could account for the conflicting results between various experiments. In chapter 6, the alga P. subcapitata was grown in culture conditions, varying in nutrient composition (ISO vs. modified ES&Walne medium), Zn concentration (0, 60 and 120 μg/L) and exposure duration (64 vs. 72 hours). This generated a set of algal stock suspensions expected to vary in Zn concentration and nutritional quality (i.e., molar C:P ratio and concentration of essential ω3-PUFA‟s). Algae were used as a diet for D. magna during a standard 21-day bioassay, using reproduction as endpoint. The development of a Generalized Linear Model demonstrated that the molar C:P ratio was the best predictor of reproduction, and that the contribution of other parameters (including dietary Zn) for a better predictability was only marginal. This illustrated the importance of the algal molar C:P ratio for predicting the reproduction of D. magna, and validation pointed out that altering dietary P levels can indeed account for the variability of reproduction between various experiments. Moreover we provided strong evidence that the assumed reproductive effects of dietary Zn at control levels in the water were rather the result of altered algal C:P ratios and a reduced dietary P availability. These results have strong implications on how to perform dietary metal toxicity experiments using living diets and how the results should be interpreted. Chapter 7 formulates general conclusions and future research perspectives, based on the research questions that have been answered in previous chapters of this dissertation. This study was the first to show ecological relevance for dietary Ni toxicity to D. magna, which can be mediated by altered resource allocation but also by a direct targeted inhibition of reproduction. We provided evidence that a reduced feeding rate is one of the major mechanisms reducing the energy supply for this test organism, whereas also a lower supply of dietary ω3-PUFA‟s cannot be ruled out. The accumulation of waterborne and dietary Ni interact synergistically, which has implications for the importance of this pathway in environmentally realistic exposures. With a view to BLM refinement, further research should elucidate the relation between the bioaccumulation and toxicity of both waterborne and dietary Ni for simultaneous exposure scenarios. An important challenge will also be to examine the importance of Ni-induced food quality shifts in natural conditions, both in a beneficial as adverse sense, on daphnids but also on other grazers. This study also provided new insights in the toxicity mechanism of Zn exposure in environments of sufficient Zn supply, where dietary Zn selectively accumulates in the reproductive tissues and waterborne Zn is more dispersive. A differentiation of pathway- specific accumulation patterns generates the enormous potential to relate bioaccumulation unambiguously to toxicity. It was also important to note that the adverse or beneficial effects of dietary Zn at control levels in the water are mainly the result of altered dietary P availabilities. This has shed new light on the toxicological implications of Zn bioaccumulation at higher levels of Zn in the water.

@phdthesis{1895310,
abstract = {Zinc (Zn) and nickel (Ni) are metals that have been increasingly mined, manufactured and employed in various applications during the past decades. This has worldwide resulted in a discharge that caused elevated levels in freshwater ecosystems. Policy makers have been urged to develop regulations to minimize the environmental risks related to Zn and Ni pollution, among other metals. Current environmental quality standards for metals, representing concentrations that pose an acceptable risk for aquatic communities, are derived on the basis of their bioavailability and not on total metal concentrations. This concept represents the principle that the toxicity of a metal is not only dependent on its total concentration, but also on the physico-chemical characteristics of the surrounding water. Bioavailability has been integrated in metal risk assessment procedures by the Biotic Ligand Model (BLM). For freshwater organisms, this model is developed from the basic assumption that the toxicity of a metal is the result of interaction with and uptake via ionoregulatory channels in the gills. However, the relatively recent discovery that dietary metals can also be toxic was expected to restrict the applicability of the BLM for several reasons. Firstly, it was assumed that this could result in an underestimation of the real exposure levels of metals, and that this in turn would end in the derivation of environmental metal standards that are not sufficiently protective. Secondly, this could also question if the mechanistic background of the BLM is still representative for the true mechanisms of metal exposure, since an extra exposure route would imply that the model assumptions are not valid anymore. Up to now, however, dietary metal exposure is still not considered explicitly in most existing metal regulations and risk assessment procedures. For various metals and aquatic species there is much uncertainty if there is relevance for this exposure route under realistic contamination levels. Also, a refinement of the BLM will only be possible until the links between dietary metal exposure and effects are better understood and quantitative methods are developed for linking metal exposure to toxicity. The freshwater flea Daphnia magna is a planktonic crustacean which occupies a central position in freshwater food webs. Its feeding and nutrition are important to both the impact on bacterial and algal populations, as well as the conversion of these micro-organisms into body mass used as food for predators. It has been extensively used as a model species for bioavailability studies and for the development of BLM\unmatched{201f}s predicting the toxicity of, among other metals, waterborne Ni and Zn. At the moment this study was started, however, the knowledge on dietary Ni and Zn toxicity was scarce. In chapter 2 we studied if there is relevance for dietary Ni toxicity to D. magna under realistic exposure levels. The green alga Pseudokirchneriella subcapitata was pre-exposed to Ni, encompassing the EC50 for chronic waterborne Ni exposure on D. magna, and used as food during a 21-day chronic bio-assay. A significant accumulation of dietborne Ni in D. magna, i.e., between 49.6 to 72.5 \ensuremath{\mu}g Ni/g dry wt, was observed when they were fed with diets containing between 85.6 and 837 \ensuremath{\mu}g Ni/g dry wt. This was paralleled by a significant reduction of reproduction (by 33.1\%) and growth (by 9.1\%). A variety of mechanisms may have been involved in these effects, including altered resource allocation or targeted reproductive inhibition. No unambiguous conclusions could be drawn, however, since these exposures were also accompanied by an altered availability of phosphorus (P) and essential omega 3 polyunsaturated fatty acids (\ensuremath{\omega}3-PUFA\unmatched{201f}s). There was no conclusive evidence that nutritional quality shifts could have contributed to the toxic effects, but it is clear that the use of diets invulnerable to metal-induced quality shifts is indispensable for a sound study of the real impact of dietary Ni exposure and its toxicity mechanisms. This problem was circumvented in chapter 3. Ni-contaminated liposomes were used as an alternative dietary Ni carrier, invulnerable to nutritional quality changes. These were produced by the hydration of phosphatidylcholine with Ni-contaminated solutions above the transition temperature of this phospholipid. Particle sizes were shown to be appropriate to be captured by the filtration apparatus of D. magna. In a first experiment, daphnids were fed with a mixture of Ni-laden liposomes and control algae as a nutrient supply. Ni bioaccumulation up to 11.9 \ensuremath{\mu}g/g dry wt after 7 days and 20.4 \ensuremath{\mu}g/g dry wt after 14 days caused a significant inhibition of reproduction and growth. It was suggested that this was mediated by a reduced nutrient absorption during the first 7 days, and additionally by a reduced algal ingestion rate beyond 7 days of exposure. A simultaneous experiment was performed, using a mixture of Ni-contaminated algae (cfr. chapter 2) and control liposomes as a diet. D. magna accumulated Ni to a similar degree as the LOEC when the algae had been pre-exposed to at least 133 \ensuremath{\mu}g Ni/L of bioavailable Ni (Ni2+), which is similar to the EC50 of waterborne Ni exposure (115 \ensuremath{\mu}g Ni2+/L). This illustrated the consequences for the use of the BLM in regulatory assessments, which may need to include the dietary toxicity pathway in this range of dissolved Ni concentrations. There was no evidence that nutritional quality shifts could have affected daphnids growth, but it is very likely that the impairment of reproduction at toxic exposure levels of Ni was also partly the result of reduced \ensuremath{\alpha}-linolenic acid levels. The integration of the dietary Ni pathway in the BLM could provide more mechanistic knowledge to predict the effects of chronic Ni exposures. It was expected that waterborne and dietary Ni bioaccumulation could be a good predictor of toxicity, but no knowledge existed on the importance of and potential interaction between both pathways during a simultaneous exposure. In chapter 4, a stable isotope tracer experiment was worked out to investigate the 7-day bioaccumulation of waterborne and dietary Ni at increasing concentrations in the water (0, 235 and 470 \ensuremath{\mu}g Ni/L) and at two pH levels (pH 6.4 and 8.2). Total Ni burdens in the daphnids evolved from 41.9 to 110.4 \ensuremath{\mu}g/g dry wt at pH 6.4 and from 26.4 to 58.5 \ensuremath{\mu}g/g dry wt at pH 8.2. Waterborne Ni uptake was significantly correlated to the concentration in the water, and was always the dominant source of uptake, contributing for at least 84\% to the total amount of Ni present in the organisms. Important to note was that the uptake of dietary Ni also increased at elevated concentrations in the water, up to 12.4 and 9.1 \ensuremath{\mu}g Ni/g dry wt at pH 6.4 and 8.2, respectively. This synergy was important from a toxicological point of view. The bioaccumulation of dietary Ni in D. magna matched the LOEC for 7-day growth at 470 \ensuremath{\mu}g Ni/L, or 115 \ensuremath{\mu}g Ni2+/L in the modified M4 medium (chapter 3). This concentration was, however, too low to generate toxic Ni burdens in algae if these would be applied as a Ni-contaminated diet during diet-only exposures. This implied that the contribution of dietary Ni uptake to the toxic effects of a combined exposure may not be neglected. The occurrence of synergy could also explain why dietary Ni accumulation did not increase with increasing pH, although the Ni concentration in the food was significantly higher. Further research should be performed to elucidate the relation between Ni uptake and toxicity during a simultaneous exposure to waterborne and dietary Ni. At the moment this research was started, the implications of dietary Zn exposure had gained far more attention than Ni. With a view to refine the BLM for chronic Zn exposure, it was investigated if an unambiguous relation could be drawn between Zn bioaccumulation and toxicity of each exposure route. In chapter 5 we investigated the hypotheses (1) that dietary Zn toxicity is the result of a selective accumulation in reproductive tissues, and (2) that waterborne and dietary Zn accumulation and toxicity are additive. D. magna was exposed to Zn following a 5 x 2 factorial design, with 5 waterborne Zn concentrations (0, 80, 170, 250 and 340 \ensuremath{\mu}g Zn/L) and 2 dietary Zn levels (50 and 496 \ensuremath{\mu}g Zn/g dry wt). Tissue-specific Zn accumulation was quantified by means of Synchrotron Radiation based Confocal X-ray Fluorescence. Waterborne Zn exposure was optimal at 80 \ensuremath{\mu}g/L, whereas organisms suffered from deficiency and toxicity at lower and higher concentrations, respectively. Dietary Zn reduced reproduction by 28\% and selectively accumulated in the eggs. These were probably provided when still present in the ovaries, which was in line with the first hypothesis. Dietary Zn was less toxic at higher Zn concentrations in the water, which could be attributed to a lower feeding rate resulting from waterborne Zn toxicity. Accordingly, no significant bioaccumulation could be detected in this concentration range. Both observations confirmed the second research hypothesis. This has obvious implications for the BLM, since the importance of dietary Zn exposures fades out at waterborne Zn concentrations that are shown to be toxic. For this reason we concluded that the BLM used in risk assessment exercises provides adequate protection against Zn exposure, and thus could take away the main concern of regulators with respect to the degree the current BLM should be refined. The complete absence of dietary Zn toxicity in the control treatment was not expected, and in contrast with previous studies dealing with dietary Zn exposure. We hypothesized that a variability of the nutritional quality could account for the conflicting results between various experiments. In chapter 6, the alga P. subcapitata was grown in culture conditions, varying in nutrient composition (ISO vs. modified ES\&Walne medium), Zn concentration (0, 60 and 120 \ensuremath{\mu}g/L) and exposure duration (64 vs. 72 hours). This generated a set of algal stock suspensions expected to vary in Zn concentration and nutritional quality (i.e., molar C:P ratio and concentration of essential \ensuremath{\omega}3-PUFA\unmatched{201f}s). Algae were used as a diet for D. magna during a standard 21-day bioassay, using reproduction as endpoint. The development of a Generalized Linear Model demonstrated that the molar C:P ratio was the best predictor of reproduction, and that the contribution of other parameters (including dietary Zn) for a better predictability was only marginal. This illustrated the importance of the algal molar C:P ratio for predicting the reproduction of D. magna, and validation pointed out that altering dietary P levels can indeed account for the variability of reproduction between various experiments. Moreover we provided strong evidence that the assumed reproductive effects of dietary Zn at control levels in the water were rather the result of altered algal C:P ratios and a reduced dietary P availability. These results have strong implications on how to perform dietary metal toxicity experiments using living diets and how the results should be interpreted. Chapter 7 formulates general conclusions and future research perspectives, based on the research questions that have been answered in previous chapters of this dissertation. This study was the first to show ecological relevance for dietary Ni toxicity to D. magna, which can be mediated by altered resource allocation but also by a direct targeted inhibition of reproduction. We provided evidence that a reduced feeding rate is one of the major mechanisms reducing the energy supply for this test organism, whereas also a lower supply of dietary \ensuremath{\omega}3-PUFA\unmatched{201f}s cannot be ruled out. The accumulation of waterborne and dietary Ni interact synergistically, which has implications for the importance of this pathway in environmentally realistic exposures. With a view to BLM refinement, further research should elucidate the relation between the bioaccumulation and toxicity of both waterborne and dietary Ni for simultaneous exposure scenarios. An important challenge will also be to examine the importance of Ni-induced food quality shifts in natural conditions, both in a beneficial as adverse sense, on daphnids but also on other grazers. This study also provided new insights in the toxicity mechanism of Zn exposure in environments of sufficient Zn supply, where dietary Zn selectively accumulates in the reproductive tissues and waterborne Zn is more dispersive. A differentiation of pathway- specific accumulation patterns generates the enormous potential to relate bioaccumulation unambiguously to toxicity. It was also important to note that the adverse or beneficial effects of dietary Zn at control levels in the water are mainly the result of altered dietary P availabilities. This has shed new light on the toxicological implications of Zn bioaccumulation at higher levels of Zn in the water.},
author = {Evens, Roel},
isbn = {9789059894532},
keyword = {Nutritional quality,Zinc,Nickel,Daphnia magna,Dietary metals},
language = {eng},
pages = {VIII, 219},
publisher = {Ghent University. Faculty of Bioscience Engineering},
school = {Ghent University},
title = {Bioavailability and toxicity of dietary nickel and zinc to the waterflea Daphnia magna},
year = {2011},
}